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Genetically determined heterogeneity of CI esterase inhibitor in patients with hereditary angioneurotic edema
Abstract
Normal human serum contains 18 +/-5 mg/100 ml of C1 esterase inhibitor (alpha-2 neuraminoglycoprotein) as estimated by immunochemical means. Of 118 patients with hereditary angioneurotic edema, the sera of 80, from 42 kindred, contained a mean concentration of 3.15 mg/100 ml or 17.5% of normal. The mean serum concentration in 35 patients in 7 other kindred was 20 mg/100 ml or 111% of normal, and 3 patients in another kindred contained over 80 mg/100 ml or greater than 400% of normal. The nonfunctional inhibitors in patients' sera of these eight kindred were identical with normal C1 esterase inhibitor by Ouchterlony analysis, but they were different from normal and from each other with respect to their electrophoretic mobility, their capacity to bind C1 esterase, and their ability to inhibit esterolysis of N-acetyl-tyrosine-ethylester.
... Two types of HAE, both inherited as autosomal dominant traits and associated with deficiency of circulating levels of functional C1 INH protein, are described. 15 Type I HAE, characterized by low plasma levels of C1 INH, accounts for 85% of cases. Type II HAE, with normal to elevated plasma levels of dysfunctional C1 INH, accounts for the remaining cases; 25% of all cases appear to arise from de novo mutations. ...
... The half-life of C1 INH is on the order of 30 to 40 hours in adults, and therefore dosing for routine prophylaxis must be fairly frequent. 15 The only C1 INH preparation FDA approved for prophylaxis is Cinryze, administered at 1000 U twice a week in adults of all sizes because there have been no dose response studies. 2,45 The 2 pC1 INH preparations (Berinert and Cinryze) are quite similar. ...
Hereditary angioedema (HAE) is a potentially life-threatening inherited disease characterized by attacks of skin swelling, severe abdominal pain, and upper airway swelling. Attacks typically begin in childhood, but the appropriate diagnosis is often missed. Attacks do not respond to epinephrine, antihistamines, or glucocorticoids. Recently, many effective drugs have been approved for treatment of adults with HAE, and the Medical Advisory Board of the HAE Patient's Association has developed and reported treatment recommendations for adults. Only 1 medication is approved for treatment of children <12 years of age, and there are no reported consensus recommendations for treatment of young children in the United States. The 11-member Medical Advisory Board, with extensive experience in the treatment of children, in concert with the leaders of the HAE Patient's Association, has developed these consensus recommendations to help in recognition, diagnosis, treatment of attacks, and prophylaxis of children with HAE.
... Clinical observations indicate that approximately 40% of the normal secretion of C1-INH is sufficient to maintain the stability of the complement system [8]. However, when C1-INH secretion falls below 30% [9][10][11], complement disorder occurs. Recent research has identified three reasons for the insufficient secretion of C1-INH. ...
Background
Hereditary angioedema (HAE) is a rare autosomal dominant genetic disease characterized by recurrent edema and a potentially fatal risk. Despite its severity, there is a notable lack of effective methods for predicting and preventing HAE attacks. This study aims to thoroughly investigate the underlying pathological mechanisms of HAE and identify potential biomarkers that could aid in its prediction and prevention.
Results
In our investigation, we have discovered a novel pathogenic variant of the SERPING1 gene, specifically c.708T > G, in a Han family affected by HAE. Our observations indicate that this variant leads to an increase in the accumulation of C1-INH within the endoplasmic reticulum (ER), resulting in the upregulation of GRP75 protein expression. This cascade of events resulted in Ca ²⁺ overload, disruption of mitochondrial structure and function, and eventually triggered apoptosis. Using siRNA to knock down GRP75 mitigates cellular calcium overload and mitochondrial damage induced by the SERPING1 mutation.
Conclusion
Based on our findings, we propose that the detection of intracellular Ca ²⁺ concentration could serve as a valuable biomarker for predicting acute attacks of HAE in patients. This discovery holds significant implications for the development of more targeted and effective strategies in the management of HAE.
... The phenomenon of elevated C1-INH levels among patients with type II HAE had been historically described in 1971 and thereafter has been mentioned in a few reports. 5 However, this is the first dedicated study to assess the diagnostic performance of high C1-INH values among patients with type II HAE and demonstrate its difference compared with non-HAE controls. We postulate that this ''paradoxical'' increase in C1-INH levels among patients with type II HAE may be a compensatory mechanism to overcome dysfunctional or inadequate C1-INH function. ...
Background
Hereditary angioedema (HAE) is a rare genetic disease. Patients with type II HAE have normal or elevated C1-inhibitor (C1-INH) levels but C1-INH protein is dysfunctional. C1-INH function requires careful sample handling and technical expertise and may account for the lack of diagnosed patients with type II HAE in resource-limited countries.
Objective
We sought to assess the diagnostic performance of elevated C1-INH levels in diagnosing type II HAE.
Methods
All patients with confirmed type II HAE in Hong Kong and India were analyzed. Diagnosis was confirmed by persistent low C1-INH function and/or pathogenic SERPING1 gene mutations. Their C1-INH levels were compared with those of matched controls.
Results
A total of 31 (14 Chinese, 17 Indian) patients with type II HAE and 31 matched controls were analyzed. Overall, 77.4% (24/31) of patients with type II HAE had elevated C1-INH levels compared with 38.7% (12 of 31) of controls (odds ratio, 2.00; 95% CI, 1.34-2.98; P = .017). C1-INH levels in patients with type II HAE were significantly higher than in controls (52.2 ± 20.0 mg/dL vs 29.1 ±3.6 mg/dL; P < .001). Findings were consistent when C1-INH values in the Chinese and Indian subgroups were analyzed separately. Receiver-operating characteristic curve demonstrated excellent performance for elevated C1-INH levels to diagnose patients with type II HAE with an area under the curve of 0.953 (95% CI, 0.941-0.992; P < .001). Positive and negative predictive values of both a low C4 and an elevated C1-INH level for patients with type II HAE were 100% and 82.9%, respectively.
Conclusions
Low C4 and elevated C1-INH levels may be considered as a screening tool for type II HAE, especially in countries where C1-INH function testing is not readily available.
... Patients with type I HAE have low levels of C1-INH protein and function; [6][7][8][9] whereas patients with type II HAE have normal C1-INH protein levels but impaired C1-INH function. 10 Patients with non-type I/II HAE have normal levels of C1-INH protein and function, collectively called normal C1-INH HAE (nC1-INH-HAE). [11][12][13] Patients with HAE have compromised quality of life due to characteristic recurrent angioedema attacks, wherein swelling (subcutaneous or submucosal) can affect 1 or multiple sites in the body including internal organs like intestine, urinary tract, and larynx. ...
Background
Hereditary angioedema (HAE), a potentially life-threatening genetic disorder due to C1 inhibitor deficiency in most cases, is characterized by sudden and/or recurrent attacks of angioedema (subcutaneous/submucosal swellings). The global World Allergy Organization (WAO)/European Academy of Allergy and Clinical Immunology (EAACI) International guideline for HAE management is comprehensive, but the implementation of this guideline may require regional adaptation considering the diversity in disease awareness, type of medical care systems, and access to diagnostics and treatment. The aim of this Delphi initiative was to build on the global guideline and provide regional adaptation to address the concerns and specific needs in the Middle East.
Methods
The Consensus panel comprised 13 experts from the Middle East (3 from the United Arab Emirates, 3 from Saudi Arabia, 2 from Lebanon, 2 from Kuwait, 2 from Oman and 1 from Qatar) who have more than 2 decades of experience in allergy and immunology and are actively involved in managing HAE patients. The process that was carried out to reach the consensus recommendation included: 1.) A systematic literature review for articles related to HAE management using Ovid MEDLINE. 2.) The development of a questionnaire by an internationally acclaimed expert, with 10 questions specific to HAE management in the Middle East. 3.) Experts received the questionnaire via email individually and their answers were recorded (email/interview). 4.) A virtual consensus meeting was organized to discuss the questionnaire, make amends if needed, vote, and achieve consensus.
Results
The questionnaire comprised 10 questions, each with 2 or more statements/recommendations on which the regional experts voted. A consensus was reached based on a 70% agreement between the participants. The key highlights include: 1) HAE experts in the Middle East emphasized the importance of a positive family history for arriving at a diagnosis of HAE. 2) The number of episodes per month or per 6-month period and severity should be used, together with other markers, to determine the need for prophylaxis. 3) Disease status should be monitored by periodic visits and the use of patient-reported outcome measures such as the angioedema activity score and the angioedema control test. 4) Attenuated androgens and tranexamic acid may be considered for long-term prophylaxis, if lanadelumab, C1-Inhibitor or berotralstat are not available.
Conclusion
This consensus recommendation may help to educate healthcare practitioners in the Middle East and unify their approach to the diagnosis and management of HAE.
... In type II C1-INH-HAE, the total concentration of C1-INH is normal or increased, owing to the simultaneous presence of the normal and of a non-functional protein. However, active C1-INH concentration is markedly decreased in both types of C1-INH-HAE (6). The decline of the plasma level of active C1-INH below a critical value (7,8) is accompanied by the activation of the plasma enzyme systems regulated by this protein. ...
C1-inhibitor (C1-INH) is an important regulator of the complement, coagulation, fibrinolytic and contact systems. The quantity of protease/C1-INH complexes in the blood is proportional to the level of the in vivo activation of these four cascade-like plasma enzyme systems. Parallel determination of C1-INH-containing activation complexes could be important to understand the regulatory role of C1-INH in diseases such as hereditary angioedema (HAE) due to C1-INH deficiency (C1-INH-HAE). We developed in-house ELISAs to measure the concentration of complexes of C1-INH formed with active proteases: C1r, C1s, MASP-1, MASP-2, plasma kallikrein, factor XIIa, factor XIa, and thrombin, as well as to determine total and functionally active C1-INH. We measured the concentration of the complexes in EDTA plasma from 6 healthy controls, from 5 with type I and 5 with type II C1-INH-HAE patients during symptom-free periods and from five patients during HAE attacks. We also assessed the concentration of these complexes in blood samples taken from one C1-INH-HAE patient during the kinetic follow-up of a HAE attack. The overall pattern of complexed C1-INH was similar in controls and C1-INH-HAE patients. C1-INH formed the highest concentration complexes with C1r and C1s. We observed higher plasma kallikrein/C1-INH complex concentration in both type I and type II C1-INH-HAE, and higher concentration of MASP-1/C1-INH, and MASP-2/C1-INH complexes in type II C1-INH-HAE patients compared to healthy controls and type I patients. Interestingly, none of the C1-INH complex concentrations changed significantly during HAE attacks. During the kinetic follow-up of an HAE attack, the concentration of plasma kallikrein/C1-INH complex was elevated at the onset of the attack. In parallel, C1r, FXIIa and FXIa complexes of C1-INH also tended to be elevated, and the changes in the concentrations of the complexes followed rather rapid kinetics. Our results suggest that the complement classical pathway plays a critical role in the metabolism of C1-INH, however, in C1-INH-HAE, contact system activation is the most significant in this respect. Due to the fast changes in the concentration of complexes, high resolution kinetic follow-up studies are needed to clarify the precise molecular background of C1-INH-HAE pathogenesis.
... 50 pLof 0.025 md/L calcium chloridewas added, and after mixing, the time required for clotting of the mixture (CT) at 37OC was measuredCinitial incubation). Each residual incubation mixture was incubated for another30 minutes with either 1 1 pL of barbital-saline buffer or 11 pL of the same buffer containing 20 Mg of purified Ci-INH, giving a final concentration of 7.7 pg of the inhibitor/100 mL of final incubation mixture (second incubation). The clotting times of these mixtures were then measured in the same manner. ...
Activated high molecular weight Hageman factor (75 Kd) and Hageman factor carboxy-terminal fragments both formed complexes with purified C1(-)-inhibitor, but the Hageman factor fragments appeared to have a higher affinity for the C1(-)-inhibitor than activated Hageman factor. Therefore, the clot-promoting activity of activated Hageman factor might be relatively unimpaired if Hageman factor fragments are also present. Normal C1(-)-inhibitor was cleaved by Hageman factor fragments. Clot-promoting activity was not generated in Hageman factor by exposure to Hageman factor fragments, nor was Hageman factor cleaved by Hageman factor fragments. When Hageman factor was cleaved by streptokinase-activated plasminogen, a 40 Kd fragment was released. In contrast to their interactions with other proteinases, which are blocked by normal C1(-)-inhibitor, Type II C1(-)-inhibitors from plasmas of affected members of eight different kindred with this form of hereditary angioneurotic edema all inhibited the specific coagulant activity of activated Hageman factor to some degree. They did not all form complexes with activated Hageman factor that were stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
... HAE type I, which accounts for 85% of cases, is caused by mutations occurring throughout the whole gene and leading to truncated or misfolded dysfunctional protein that is not secreted efficiently, thus the C1INH plasma level is decreased. HAE type II, which accounts for 15% of cases, is usually caused by mutations in a reactive center loop coding part of exon 8, leading to a mutant protein that is secreted but dysfunctional (Cugno et al., 2009;Rosen et al., 1971). In both HAE types, kallikrein-kinin system activation is dysregulated, which leads to an excessive generation of bradykinin, the major swelling mediator in HAE (Joseph et al., 2008;Nussberger et al., 1998). ...
Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3' splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3' ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3' ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3' ss seems to be co-regulated with the authentic 3' ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.
... These 2 situations account for the genetic variants of HAE described by Rosen et al 14 : type I, in which there are dramatically reduced levels of C1-INH protein, 13 and type II, in which C1-INH protein is synthesized but fails to function adequately. C4 levels are typically low in HAE types I and II. ...
Importance:
Hereditary angioedema (HAE) is a rare but severe disease, with high risk of death, and attacks have been associated to high estrogen levels. Polycystic ovary syndrome (PCOS) is a common hyperandrogenic condition, which is frequently treated with combined oral contraceptives.
Objective:
The aim of this study was to describe 2 clinical cases of young women diagnosed as having PCOS who developed HAE attacks after the introduction of combined estrogen-progestin pills to treat PCOS symptoms.
Evidence acquisition:
Literature review of sex hormones' role in genesis of HAE attacks and possible mechanisms involved.
Results:
In the cases reported, after initiation of combined contraceptives, patients presented with facial swelling with airway involvement (laryngeal edema) and abdominal pain. They had a familial history of angioedema and normal C1 inhibitor (C1-INH) levels, leading to the diagnosis of HAE with normal C1-INH (HAEnC1-INH) or HAE type III. After suspension of exogenous estrogen, patients remained asymptomatic from HAE.
Conclusions and relevance:
HAEnC1-INH is an estrogen-dependent form of HAE. It is well established that exogenous estrogen triggers attacks of all types of HAE. However, this is the first description of the association between PCOS and HAE, in which PCOS could be masking HAE symptoms. We propose that PCOS might have a protective role regarding HAE attacks, because of its particular hormonal features, that is, hyperandrogenism and relative stable levels of estradiol. The use of combined estrogen-progestin compounds in women with PCOS and HAE must be avoided, and treatment must be individualized.
... Распространенность заболевания точно неизвестна, приблизительно встречается с частотой 1 : 50 000-100 000, без различий для разных этнических групп [5,6]. Несмотря на редкую встречаемость, НАО представляет серьезную проблему в практической медицине, в связи с тяжестью обострений и высокой вероятностью развития тяжелых осложнений, которые в некоторых случаях приводят к летальному исходу [7]. Изучение патогенеза наследственных форм ангиоотеков позволяет проводить не только дифференциальную диагностику, но и верифицировать НАО на досимптомной (доклинической) стадии. ...
... Two phenotypic variants were described [32,33]. Type I (HAE-I) is the most common (85%), characterized by a quantitative decrease of C1-INH, which results in a decrease in functional activity; type II (HAE-II) (15%) is characterized by normal or elevated levels of dysfunctional C1-INH. ...
The “complement system” is one of the effector pathways of the immune system against microorganisms and tumor cells. The complement system can be activated through three major pathways: classical, lectin, and alternative. The sequential activation through the generation of complex enzymes from inactive zymogens produces a cascade in which a capable enzyme generates a large number of active downstream molecules.
C1 inhibitor (C1‐INH) is a serine protease inhibitor (serpin) that regulates the following closely interrelated proteolytic pathways: complement system, coagulation system, contact system, and fibrinolysis system. The absence or malfunction of C1‐INH results in the presence of attacks of angioedema (AE) due to uncontrolled activation of the contact system, with the generation of bradykinin (BK), a vasoactive peptide released from high‐molecular‐weight kininogen (HMWK). Some drugs that inhibit the catabolism of BK have been implicated in the development of AE. These include angiotensin‐converting enzyme inhibitors (ACEIs), dipeptidyl peptidase IV (DPP‐IV) inhibitors, aminopeptidase P (APP) inhibitors, and neutral endopeptidase (NEP) inhibitors.
We describe in this chapter the biochemistry pathways implicated in the pathophysiology of bradykininergic angioedema (BK‐AE) and the role of the complement system in the prototype of BK‐AE, in hereditary angioedema with C1‐INH deficiency (C1‐INH‐HAE), and also in acquired angioedema with C1‐INH deficiency (C1‐INH‐AAE).
... These patients have normal antigenic levels but abnormal C1-INH function. 7,8 Under physiological conditions, C1-INH regulates the activities of four interlinked proteolytic enzyme cascades, namely, the complement, contact (kallikrein-kinin), fibrinolytic, and coagulation pathways ( Figure 1). In particular, C1-INH is the primary inhibitor of the kallikrein-kinin system via inactivation of activated factor XII (factor XIIa) and kallikrein. ...
Hereditary angioedema (HAE) is a rare genetic disease characterized by episodic subcutaneous or submucosal swelling. The primary cause for the most common form of HAE is a deficiency in functional C1 esterase inhibitor (C1-INH). The swelling caused by HAE can be painful, disfiguring, and life-threatening. It reduces daily function and compromises the quality of life of affected individuals and their caregivers. Among different treatment strategies, replacement with C1-INH concentrates is employed for on-demand treatment of acute attacks and long-term prophylaxis. Three human plasma-derived C1-INH preparations are approved for HAE treatment in the US, the European Union, or both regions: Cinryze®, Berinert®, and Cetor®; however, only Cinryze is approved for long-term prophylaxis. Postmarketing studies have shown that home therapy (self-administered or administered by a caregiver) is a convenient and safe option preferred by many HAE patients. In this review, we summarize the role of self-administered plasma-derived C1-INH concentrate therapy with Cinryze at home in the prophylaxis of HAE.
... This type of HAE is characterized by normal plasma antigenic levels of C1 inhibitor but decreased functional levels of the plasma C1 inhibitor. 20,24 Most of SERPING1 mutations associated with type II HAE involve residues at or near the active site on the reactive mobile loop that result in a mutant C1 inhibitor protein that is secreted but is dysfunctional. 25 In both type I and type II HAE, the low functional level of C1 inhibitor results in diminished regulation of the complement and contact systems. ...
Hereditary angioedema (HAE) causes recurrent episodes of angioedema that may be very severe and are frequently associated with significant morbidity and even mortality. Understanding the pathophysiology of this disease is crucial for proper diagnosis and management of these patients. HAE is caused by mutations in the SERPING1 gene that result in decreased plasma levels of functional C1 inhibitor. A large number of different mutations have been described that result in HAE. About 15% of patients have a mutation at or near the active site of the reactive mobile loop, resulting in a protein that lacks functional activity (type II HAE). Type I HAE is caused by a diverse range of mutations, some of which cause the nascent protein to misfold and thus to be unable to enter the secretory pathway. The primary mediator of swelling in HAE is bradykinin, a product of the plasma contact system. Bradykinin induces increased vascular permeability by activating the bradykinin B2 receptor, which results in phosphorylation of vascular endothelial cadherin. The regulation of both the bradykinin B2 receptor and peptidases that degrade bradykinin may influence HAE disease severity. HAE results from mutations in the SERPING1 gene that lead to a loss of functional C1 inhibitor. Attacks of angioedema result from generation of bradykinin, which acts on bradykinin B2 receptors to enhance vascular permeability.
... Hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-C1-INH) is a primary immunodeficiency [1]. It is transmitted in an autosomal dominant manner, and 2 phenotypic variants have been described [2]: HAE-C1-INH type I, which is due to a quantitative decrease in levels of C1esterase inhibitor (C1-INH) and is the more frequent (85%), and HAE-C1-INH type II (15%), which is caused by a deficit in the functioning of C1-INH [3]. HAE-C1-INH is a rare disease, with an estimated prevalence of between 1 in 10 000 and 1 in 50 000 individuals [4] and a minimal prevalence of 1.09 cases per 100 000 inhabitants in Spain [5]. ...
Background: Hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-C1-INH) is a life-threatening disease. Objectives: To describe the clinical characteristics and management of patients with HAE-C1-INH during routine clinical practice. Methods: An observational, retrospective study was performed in patients with HAE-C1-INH. Demographic, clinical, and analytical data were collected from 2 periods: period A (October 2009-September 2010) and period B (October 2007-September 2009). Results: We studied 112 patients with HAE-C1-INH (57.1% females). Age at onset of symptoms was 14.4 years (lower in patients who had experienced attacks in the previous year). In period B (n=87), 62.1% of patients presented at least 1 edema attack (median, 3.5 attacks/ patient/2 years), and 19.1% of attacks were treated. In period A (n=77), 58.4% of patients were on maintenance therapy. Stanozolol was the most widely used drug (48.9%), with a mean weekly dose of 6.7 mg. At least 1 attack was recorded in 72.7% of patients (median, 3.0 attacks/patient/year), and 31.5% of the attacks were treated. Treatment of acute attacks increased by 12.4%. Conclusion: Age at onset of symptoms is associated with clinical expression of disease. The higher age at onset of symptoms, the fewer number of attacks per patient and year, and the lower dose of attenuated androgens necessary to control the disease than in other series lead us to hypothesize that HAE-C1-INH could have a less severe expression in Spain. Acute attacks seem to be treated increasingly often.
... C1INH has a long history of use as a drug; indeed, it can claim to be the 'first-in-man' complement drug and the first of the natural inhibitors to be recognized as a potential therapeutic. More than 40 years ago, patients with HAE were shown to be deficient in C1INH and to respond to plasma replacement, provoking efforts to purify C1INH for treatment of acute attacks 59,61 . Remarkable successes in these early studies rapidly led to the adoption of plasma-derived C1INH as the standard of care for acute episodes in HAE. ...
The complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. Inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. Complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. Drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. Only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. In this Review, we describe the history, current landscape and future directions for anti-complement therapies.
... However, in rare cases, the C4 level can be normal between attacks [32]. Measurement of decreased antigenic and functional C1 inhibitor levels confirms the diagnosis of HAE and differentiates between type I and type II [33,34]. Differentiation from an acquired C1-inhibitor deficiency causing acquired angioedema (AAE) can be made by measurement of C1q, as C1q is low in AAE. ...
The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed. This article is protected by copyright. All rights reserved.
... 4,5 On the basis of the C1-Inh levels in plasma, there are 2 HAE subtypes: patients with HAE type I (approximately 80% to 85%) have low antigenic and functional C1-Inh levels, whereas patients with HAE type II (approximately 15% to 20%) have normal or increased antigenic levels but low C1-Inh function. 6,7 The C1NH mutations causing HAE type II are usually missense mutations located within or around the reactive center and the nearby hinge region, whereas the mutations causing HAE type I are very heterogeneous and are distributed over the entire gene (see the C1NH mutation database at www.hae.biomembrane.hu). As an autosomal dominant disease, patients with HAE are heterozygous, and to date, only one family with a homozygous mutation affecting the promoter region has been reported. ...
Background:
Patients with hereditary angioedema (HAE) experience recurrent, sometimes life-threatening, attacks of edema. It is a rare genetic disorder characterized by genetic and clinical heterogenicity. Most cases are caused by genetic variants in the SERPING1 gene leading to plasma deficiency of the encoded protein C1 inhibitor (C1INH). More than 500 different HAE-causing variants have been identified in the SERPING1 gene, but the disease mechanisms by which they result in pathologically low C1INH plasma levels remain largely unknown.
Objectives:
The aim is to describe trans-inhibitory effects of full-length or near full-length C1INH encoded by 28 disease-associated SERPING1 variants.
Methods:
HeLa cells are transfected with expression constructs encoding the studied SERPING1 variants. Extensive and comparative studies of C1INH expression, secretion, functionality, and intracellular localisation are carried out.
Results:
Our findings characterize functional properties of a subset of SERPING1 variants allowing the examined variants to be subdivided into five different clusters, each containing variants sharing specific molecular characteristics. For all variants, except two, we find that co-expression of mutant and normal C1INH negatively impacts the overall capacity to target proteases. Strikingly, for a subset of variants, intracellular formation of C1INH foci is detectable only in heterozygous configurations enabling simultaneous expression of normal and mutant C1INH.
Conclusions:
We provide a functional classification of SERPING1 gene variants suggesting that different SERPING1 variants drive the pathogenicity through different and in some cases overlapping molecular disease mechanisms. For a subset of gene variants, our data define some types of C1INH-HAE as serpinopathies driven by dominant-negative disease mechanisms.
Genetic deficiencies of almost all the 20 proteins of the complement system have been described. They are all inherited as autosomal recessive phenomena with the exception of properdin (X-linkage) and CI 1NH (autosomal dominant). The deficiencies readily fall into five categories that are distinctive but not without overlap. They present with angio-oedema (CI 1NH); immune complex disease (Clq, Or, C4, C2); susceptibility to pyogens (C3, factor H, factor I) or Neisseria (C5, C6, C7, C8, properdin). C9-deficient individuals appear to be mostly asymptomatic. Rapid progress is being made in understanding the molecular biology of these deficient states.
Angioedema is a rare adverse effect of the commonly used angiotensin converting enzyme inhibitors (ACEi) and is reported to occur with a prevalence of 0.1 – 0.7%. Although most ACEi‐induced angioedema (ACEi‐A) cases are mild, severe cases requiring intensive care and even resulting in death have been reported in the literature. The mechanisms underlying ACEi–A are not yet fully understood, but bradykinin and/or substance P accumulation resulting from inhibition of ACE, is believed to play a crucial role. ACEi‐A occurs at variable frequencies across different racial groups, suggesting a genetic association to the development of ACEi‐A. To date, one genome wide association study and several candidate gene studies have been published on the association of genetic variation with ACEi‐A. Genetic associations reported have been attributed to several distinct mechanisms: (1) genes coding for alternative enzymes responsible for the degradation of bradykinin and/or substance P in the diminution of ACE activity (2) ACE gene function, (3) bradykinin receptor genes, (4) genes implicated in immune and inflammation regulation, and (5) genes in the fibrinolytic and coagulation pathway. Despite several plausible genetic associations, there are currently no genetic variants with sufficient effect to be clinically useful. The low incidence of ACEi‐A suggests that a combination of genomic approaches with the capability to detect potentially important variants might be required to shed light on the mechanism of this adverse reaction. Additionally, many non‐genetic risk factors associated with ACEi‐A suggest the potential contribution of epigenetic dysregulation.
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Remarkable progress in understanding the pathophysiology and underlying mechanisms of hereditary angioedema has led to the development of effective treatment for this disorder. Progress in three separate areas has catalyzed our understanding of hereditary angioedema. The first is the recognition that HAE type I and type II result from a deficiency in the plasma level of functional C1 inhibitor. This observation has led to a detailed understanding of the SERPING1 mutations responsible for this deficiency as well as the molecular regulation of C1 inhibitor expression and function. The second is that the fundamental cause of swelling is enhanced contact system activation leading to increased generation of bradykinin. Substantial progress has been made in defining the parameters regulating bradykinin generation and catabolism as well as the receptors that transduce the biologic effects of kinins. The third is the understanding that tissue swelling in hereditary angioedema primarily involves the function of endothelial cell adherens junctions. This knowledge is driving increased attention to the role of endothelial biology in determining disease activity in hereditary angioedema. While there has been considerable progress made, large gaps still remain in our knowledge. Important areas that remain poorly understood include the factors that lead to very low plasma functional C1 inhibitor levels, the triggers of contact system activation in hereditary angioedema, and the role of the bradykinin B1 receptor. The phenotypic variability of hereditary angioedema has been extensively documented but never understood. The mechanisms discussed in this chapter likely contribute to this variability. Future progress in understanding these mechanisms should provide new means to improve the diagnosis and treatment of hereditary angioedema.
The term “swelling” has been used in the old scriptures to illustrate a change of normal figure and, as such, an expression of illness. It should be noted that in ancient times, human diseases were very often regarded a punishment from God. Hence, it is not surprising that one of the oldest tests for infidelity involved swelling as an inflicted punishment. The great Greek physician Hippocrates (377–460 BC), considered one of the most outstanding figures in the history of medicine and “Father of the Western Medicine,” already used the term oídēma to describe swelling of organs. It took many centuries later until the first description of angioedema as a distinct medical entity was minted by Quinke in 1882. The historical progression in angioedema research has been characterized by intermittent “leaps” in interest and scientific achievements. As an example, it took 75 years from the accurate description of hereditary angioedema (HAE) by Osler (1888), until a group of researchers headed by Donaldson (1963) disclosed the central role of C1 inhibitor in angioedema pathophysiology. What followed was a result of a collective effort by many researchers and scientific groups who were able to elucidate the intricate connections between the implicated biochemical pathways. Still, scientific progress was hardly translated into effective therapy, and another 45 years had to elapse until the renewed interest in HAE was boosted by studies on the efficacy and safety of novel therapies about 10 years ago. In the twenty-first century, HAE ceased to be an “orphan disease” and its future is far more optimistic. It is better managed now by specialized angioedema centers, harmonized clinical guidelines, educational programs, laboratory services, and continued basic and clinical research. Patient associations worldwide are offering support and guidance, and governments and healthcare systems are gradually addressing patient and family needs.
The complement system constitutes an important effector of host defenses but may also serve as a mediator of immunopathological events. Genetically determined human complement deficiencies were first recognized about 25 years ago. These observations, coupled with advances in protein chemistry and molecular biological studies, have stimulated great interest in the genetic disorders of the complement system. As a consequence, better understanding of the biological function and role of complement in human disease has been facilitated.
Alterations in the complement (C) system in human pathology have been recognized for many years, but only within recent years have measurements of serum C components become clinically available. In view of the apparent different biological properties of each component of the C system (Ruddy et al., 1972a; Müller-Eberhard, 1975), a proper understanding of the role of C in disease requires an analysis not only of the whole hemolytic C, but also of its components. Immunochemical measurements using monospecific antibodies against C proteins are available, but fail to distinguish between native proteins and those that have been rendered inactive either because they have participated in the C reaction sequence or because they have been incorrectly synthesized de novo. Stoichiometric hemolytic titrations provide sensitive assessment of the functional activity of the individual C component.
This chapter discusses the use of radiolabeled bacterial Fc-binding proteins as tracers for soluble antigens. The practical considerations in using radiolabeled tracers for measuring antigen–antibody complexes require that the labeled tracer bound to the antigen–antibody complex can be separated easily from the unbound tracer. This can be achieved by first immobilizing the antigen, allowing the antibody to react, and then monitoring the immunoglobulin associated with the immobilized antigen–antibody complex with the labeled tracer. Thus, most immunoassays at present use some form of immobilized antigen. Radioiodinated bacterial Fc-binding proteins from a number of sources have been shown to be suitable tracers in radioimmunoassays. The appropriate bacterial Fc-binding protein to use as a radiolabeled tracer in a specific assay depends upon a number of factors. Of primary concern is the reactivity of the bacterial Fc-binding protein with the species of immunoglobulin used as the specific antiserum in the assay. The procedure for optimizing the reagents is a necessary first step in developing any competitive-binding radioimmunoassay. The optimal reaction conditions may vary for antibodies from different species or for certain antigen–antibody pairs.
In der dermatologischen Fachzeitschrift „Der Hautarzt“ findet sich 1972 der Artikel eines Mainzer Autors mit dem Thema „Das Komplement: Vermittlungssystem für humorale Abwehrleistung und allergische Entzündung“ [40]. Beim Lesen dieser nur fast 10 Jahre alten Arbeit läßt sich erfreulicherweise feststellen, daß sich praktisch nichts von der damaligen Schilderung des Komplementsystems in der Zwischenzeit als falsch erwiesen hat. Das damals noch schlanke Bäumchen der Komplementforschung hat aber nicht nur Jahresringe angesetzt, sondern vor allem mächtige Äste getrieben, wie z. B. die antikörperunabhängige Aktivierung des Systems, den Nachweis von Komplementrezeptoren auf verschiedenen Zellklassen und eine differenzierte Komplementgenetik.
Proteolytic enzymes were among the first known enzymes. At the beginning of this century biologists became aware of the involvement of these enzymes in biological control mechanisms. Evidently these early investigators were primarily concerned with enzymes such as trypsin achieving extensive digestion of their protein substrates. Later the extra- and intra-cellular location of proteolytic enzymes was stated and in more recent years the fundamental role of limited proteolysis achieving the activation or modification of a substrate molecule, enabling it to take its place in a reaction cascade, became more and more the center of interest in many laboratories. Obviously there are too many control mechanisms in intra- and extracellular proteolysis to be reviewed here, and this paper will only describe the control of some proteolytic activities in mammalian plasma by eight of the actually known antiproteases. These inhibitors interfere mainly with the proteolytic activities of clotting components, of those enzymes controlling blood pressure and those achieving fibrinolysis. But we have also to take into account the complement system and the proteases liberated by blood cells, namely platelets and leukocytes, as these latter enzymes are able to activate or destroy factors belonging to the before-mentioned systems.
C1̄ inhibitor is a highly glycosylated member of the serpin gene family. Mutations in dysfunctional C1̄ inhibitor proteins cluster in two regions. A high rate of mutational change of the reactive site P1 residue may be explained by the presence of a CpG dimer in its codon. Mutations in the highly conserved region just N-terminal to the reactive site change dysfunctional C1̄ inhibitor proteins to substrates. The structure and function of serpins are discussed on the basis of these mutational studies, sequence alignments and the known tertiary structures of α1-antitrypsin and plakalbumin.
The complement system (C) is composed of group of plasma proteins which under certain conditions react with each other sequentially, resulting in the formation of highly specialized proteases. By their action multimolecular complexes are assembled, and when these events occur on the surface of some cells, the membrane function is impaired and the cell will lyse. Fragments and complexes appearing on activation of the complement system participate in other important biological activities such as chemotaxis, opsonization and in histamin release from mast cells and basophilic granulocytes.
Urticaria and angioedema are important medical problems in patients of all ages. The pathogenesis always involves blood vessels and mast cells, either in the superficial dermis or in the deep dermis or hypodermis. The pathophysiology can be explained by vasodilatation and increased vascular permeability resulting from interaction between the causative agent and the vasculature or the perivascular mast cells, with subsequent release of vasoactive mediators.
A clinical classification system of causative agents has been developed for use by clinicians in the evaluation of previously undiagnosed patients with urticaria or angioedema (see Table 4). The seven well-recognized general causes of urticaria/angioedema are physical stimuli, contact agents, ingestants, inhalants, infections, systemic diseases, and psychogenic factors. Contact agents, inhalants, infections, and psychogenic factors do not seem to be common causes of urticaria/angioedema in patients of any age, whereas physical stimuli can be important causative agents, especially in younger patients. The importance of systemic diseases increases as the age of the patients increases. Among the systemic diseases, endocrine system disorders—especially diabetes mellitus and thyroid disorders—seem to be the most common causative agents; malignancies can be associated with urticaria/angioedema, but relatively infrequently. Finally, ingestants are important causes of urticaria and angioedema in patients of all ages. Dietary agents continue to induce these conditions in a significant number of elderly patients. In addition, medications such as β-lactam antibiotics, NSAIDs, and ACE inhibitors are of special importance.
Systematic evaluation, including a detailed medical history, physical examination, and appropriate laboratory tests, with emphasis on systemic diseases, dietary factors, and medications, will identify the etiologic agent(s) in at least 50% of elderly patients.
Our recognition of the association between C1INH deficiency and autoimmunity has increased significantly in the past decade. Individuals with HAE have a number of immunoregulatory abnormalities and appear at increased risk for expressing autoantibodies or developing autoimmune disease. The basis for this association is not clearly understood. Hereditary angioedema is not causally related to either autoimmune diseases or immunoregulatory abnormalities. It also is not HLA-linked. It appears that the relationship between autoimmunity and HAE is mediated by the products of complement or contact system activation. Further elucidation of the mechanisms responsible for this association may contribute to an improved understanding of the development of autoimmunity.
The link between autoimmunity and the development of acquired C1INH deficiency is of particular interest. It has become evident that a substantial number of acquired C1INH-deficiency patients have anti-C1INH autoantibodies that appear to be responsible for the development of the C1INH deficiency. Anti-C1INH autoantibodies interfere with normal interactions between C1INH and proteases, leading to increased C1INH catabolism. Contrary to previous expectations, these patients do not appear to be at increased risk for an underlying malignancy. Other patients with acquired C1INH deficiency, with or without an underlying malignancy, may have autoantibodies of different specificities that contribute to the increased C1INH catabolism by forming immune complexes and activating complement. The recognition of autoimmune processes in the development of acquired C1INH deficiency also alters the range of therapeutic approaches that need to be considered in treating this disease.
Studies on the biological activity of complement have revealed that the exposure of neoepitopes in complement components and the subsequent removal of activated peptides are typical mechanisms of complement activation. It has been reported that the generation of binding regions and the appearance of hidden epitopes can result in an autoantibody response. Examples of this disregulation of the immune system are autoantibodies against degradation products of C3 and C4, the so-called ‘immunoconglutinins’ and autoantibodies against the classical or alternative pathway convertases.
The hereditary transmission of angioneurotic oedema was documented as early as 1888 by the remarkable pedigree investigations of W. Osier1. His work and subsequent family studies led to the recognition of the autosomal dominant mode of transmission of the disease2.
This chapter provides an overview of bradykinin (BK) formation in allergic diseases. The plasma kinin-forming system consists of three essential plasma proteins, which interact in a complex fashion once bound to certain negatively charged surfaces or macromolecular complexes. These are coagulation Factor XII, prekallikrein and high-molecular-weight kininogen (HMWK). Once Factor XII is activated to Factor XIIa, it converts plasma prekallikrein to kallikrein (KLK) and KLK digests HMWK to liberate BK. Factor XIIa also converts coagulation Factor XI to Factor XIa to continue the intrinsic coagulation cascade. The interaction of all four of these proteins to initiate blood clotting is known as “contact activation”; thus, the formation of BK is a cleavage product of the initiating step of this cascade. The pathogenesis of hereditary angioedema (HAE) suggests liberation of a kinin that has variously been considered a product of the second component of complement or produced by contact activation. If C1 inhibitor is either absent (Type I HAE) or dysfunctional (Type II HAE), there is insufficient inhibition of all the activated forms of Factor XII, KLK or activated C1. Production of BK is markedly augmented under these conditions. The addition of dextran sulfate at concentrations insufficient to activate normal plasma leads to complete digestion of HMWK in HAE plasma within a few minutes. Thus, seemingly insignificant traumas or infections may be sufficient to initiate an attack in such patients.
The bactericidal activity of serum was the key observation in the discovery of the complement system, and, accordingly, it was originally defined by that function. Even though the definition was later on for technical reasons based on the reactivity in a hemolytic system, the bactericidal activity was generally accepted as its main function in vivo. The killing of bacteria by complement was seen as a most important, or even as the decisive, factor in host defense against bacterial infections. Understandably, then, it was a great surprise when in 1919 [30] a strain of guinea pigs was discovered which was deficient in complement. Hemolysis in vitro was totally lacking in some animal sera and was greatly reduced in the others. Lytic activity was restored with heat-inactivated serum [21] but not by serum treated with yeast cells or with cobra venom factor [8]. The deficiency was ascribed to a defect in what at that time was called C3. When previously immunized, the animals were able to cope normally with bacterial infections. Their nonspecific resistance to infection was, however, greatly impaired and the opsonic capacity of their serum in vitro was reduced to about one-half of normal levels [30]. Since the strain was lost in the late 1920s, long before the then C3 was recognized to comprise the factors C3, C5, C6, C7, C8, and C9, it now is only possible to speculate on which of the C3–C9 components had specifically been missing. In any case, the loss seemed to support the theories of those scientists who regarded complement as a life-preserving system.
The complement system consists of some twenty serum proteins that interact with one another in highly specific ways as part of host defence and of the inflammatory reaction, primarily in response to infection and in the clearance of immune complexes1. The system appears also to participate in the pathophysiology of ‘autoimmune’ diseases such as systemic lupus erythematosus and glomerulonephritis2. In the latter disorders, lowering of the serum concentration of individual complement proteins, deposition of complement proteins in affected tissues, particularly renal glomeruli, and the presence in the circulation of complement protein fragments are taken as evidence of complement activation in vivo, probably by immune complexes. The concentration of some complement proteins is also lowered in hepatic diseases. Because many complement proteins are produced primarily in the liver, it has been suggested that failure of the protein-synthesising capacity of this organ may contribute to or be largely responsible for lowered complement in liver diseases. Many, but not all, complement proteins are acute-phase reactants and rise in serum concentration following tissue necrosis or inflammation. Finally, there are inherited deficiency states, mostly rare, for almost all complement proteins. For inherited deficiencies of complement components themselves, it is, by and large, only the affected protein which is markedly decreased in concentration or absent. On the other hand, deficiency states of control proteins produce complex patterns of reductions in the concentrations of certain individual proteins.
Hereditary angio-oedema is defined clinically by episodic attacks of painless non-pruritic swelling of the external surfaces of the body (Figure 8.1) and/or the gastrointestinal tract that affects multiple generations of a family1-4. In the American literature, credit for the original description is often given to William Osier who described a family with the disease affecting members of four generations5. The autosomal dominant nature of the inheritance pattern (Figure 8.2) was remarked upon in this early study. In fact, the initial observations of this disease are older; in Europe, the disease is often known as Quincke’s oedema6 and there are older descriptions. As originally noted, the disease is defined by episodic attacks of non-pruritic oedema that affect any external surface of the body as well as by attacks of severe abdominal pain1-4. The attacks usually have a duration of one to three days, occurring with no clear pattern. Although clinically the disease is characterized by the occurrence of episodes of oedema of the deeper tissues of the dermis leading to marked local swelling, other organs can be affected. The older literature suggests that many organs can be so affected and there are reported cases of angio-oedema of cerebral tissue leading to episodic hemiparesis as well as angio-oedema of many other organs leading to dysfunction3. The original description of Quincke includes such an attack6. In fact, those who have followed large numbers of patients with biochemically proven hereditary angio-oedema have not been struck by the frequent attacks of angio-oedema of internal organs with the single exception of the gastrointestinal tract.
We report a case of angioedema in the tongue and oral floor potentially induced by angiotensin II receptor blockers (ARB). A 77-year-old woman with a history of hypertension was treated with an ARB, and edematous lesions in the tongue and oral floor suddenly occurred about one week after the start of the treatment. She visited an emergency hospital and was prescribed serrapeptase, a complex of betamethasone and chlorpheniramine maleate, and epinastine hydrochloride. But since the edema was poorly controlled, she was referred to our department. The ARB was strongly suspected to have caused the angioedema, so we stopped the ARB that she was taking. Edema disappeared one day after the onset of symptoms, and there has been no recurrence of symptoms after the termination of ARB treatment.
Three biochemical pathways of complement activation are known. The first pathway, defined a century ago, is termed the classical pathway. This pathway is usually activated by antibody, and was identified because of its ability to kill antibody sensitized bacteria. A second pathway, now termed the alternative pathway, was first observed in the 1950s and was studied in detail in the 1970s and 1980s. A third pathway, the lectin pathway, described in the past two decades, is still being defined in detail. The latter two pathways are phylogenetically older than the classical pathway, and do not require antibody to function. They are found in organisms as primitive as sea squirts. All three pathways proceed through a series of protein interactions, discussed in this chapter, to the activation and binding of a plasma protein C3, which is central to all three pathways. The pathways then proceed together through the binding of an additional series of proteins to the lytic and inflammation promoting steps in complement action. Deficiencies of nearly every component have been described. The clinical features are diverse, but cluster into features that align with the known functions of complement: prevention of infection, disposal of apoptotic cells and immune complexes, and protection of endothelial surfaces.
The discovery of X-linked agammaglobulinaemia in 1952 was only a prelude to the discovery of a large number of deficiency states in the immunological systems. At present, almost all the well-defined immunodeficiencies in man and animals have a genetic basis. Despite this fact, it is curious that very few examples of structural gene defects have come to light. Most of the immunodeficiency diseases appear to result from the failure of some regulatory process, and a disproportionate number of them are X linked.
We report the case of an 8-years-old child with hereditary angioedema who required oral surgery.
Hereditary angioedema (HAE) is a rare autosomal dominant hereditary disease caused by a functional disorder of C1 inhibitor (C1-INH) or a low level of C1- INH protein. HAE is characterized by repeated localized edema of submucocutaneous tissues. Attacks are caused by trauma, drugs, infection, or mental stress. Dental treatment and oral surgery in patients with HAE can trigger fatal laryngeal edema.
To prevent angioedema attacks, we injected C1-INH before surgery in a child with HAE. She underwent tooth extraction and cystectomy of the left side of the mandible under general anestethia. No HAE-related complications occurred during or after surgery, and perioperative management went well.
The capacity of plasma and serum to inhibit proteinases was recognized nearly a century ago and this phenomenon has been studied extensively since. It is not the purpose of this chapter to discuss the early work in any detail; this has been done elsewhere.1 In the last decade, the chemical identity of several plasma proteinase inhibitors has been established, leading to a great increase in knowledge of the activities and functions of these substances. Comprehensive accounts of this information are available in several monographs that have appeared in recent years.2–4 We will emphasize recent advances that promise to provide a clearer understanding of the mechanisms of action and function of proteinase inhibitors.
Introduction. The nine components1 that comprise the classical pathway constitute a family of proteins whose functions can be divided into three phases: recognition, activation, and attack. Most early work in the field focused on defining the functional activities of the various components, leading to our present concept of their reaction sequence. More recently, as modern biochemical techniques have been used to study these proteins, the physicochemical basis for their activities has begun to be elucidated.
From the original meaning of “complement” as the heat-labile substance present in serum and required for the killing of certain bacteria, the term “complement system” has evolved to denote a special group of normal serum proteins which interact sequentially to effect a variety of inflammatory events, including bacterial lysis. The term “properdin” was originally used to signify that substance in normal human serum which is required for zymosan to destroy the third component of complement, but has now come to designate a single protein among the four or five required for the zymosan-induced reaction. “Properdin system” is used to denote the entire sequence of serum proteins which participate in this reaction.
This chapter emphasizes the potential applications of studies of complement biosynthesis and presents a review of the historical aspects of work on this problem. The methods for studies of complement synthesis include tissue culture conditions (media and cells), assay systems, biosynthesis in vivo, and a criteria for establishing that a given cell or tissue is a site of synthesis of a complement protein. The sites of synthesis of the individual complement proteins are described based on the biosynthesis of C1, C2, C4, C3, C5 and C6, C7, C8, C9, and complement–associated proteins. The approaches to study fetal synthesis of complement are (1) synthesis of specific complement components in vitro by isolated fetal tissues; (2) demonstration of a maternal-fetal discordance of genetic type in those complement proteins exhibiting genetic polymorphisms; and (3) a method showing the presence of a complement component in sera of fetuses borne by genetically deficient mothers. Most of the complement proteins are synthesized early in fetal life, and in several species (man, rodents, and ungulates) the placenta is an effective barrier to passage of complement either from or to the fetal circulation. The chapter describes the genetically determined complement deficiencies such as C4 deficiency, C5 deficiency, C2 deficiency, and deficiency of C1 inhibitor in man and experimental animals. The advantage of using complement as a model system for genetic studies is the ease of detecting the gene product, the large number of well-characterized genetic abnormalities, and the availability of cell lines capable of synthesizing individual complement proteins. Finally, the chapter discusses the nongenetic control of complement biosynthesis.
Hereditary angioedema (HAE) is characterised by episodic swelling of the extremities, face, larynx and recurrent abdominal pain, which can mimic the acute abdomen. Trauma of the larynx may result in acute airway obstruction. The management of emergency anaesthesia for Caesarean section of a patient with documented HAE is described and the special problems presented discussed. The methods of prophylaxis available are considered and the use of fresh frozen plasma advocated.
Although variants of sialic acid-free alpha(1)-acid glycoprotein have been described in human beings, the mode of inheritance of these types has not been reported previously. With the use of a new technique of immunofixation after agarose gel electrophoresis of neuraminidase-treated whole serum, the present study demonstrates that the types of alpha(1)-acid glycoprotein variants in family members are consistent with inheritance as autosomal traits with codominant expression. Gene frequencies have been determined for several ethnic groups. Of a total of 11 maternal-cord serum pairs, seven were discordant types, indicating that the fetus synthesizes alpha(1)-acid glycoprotein and confirming a previous report that there is no significant transplacental passage of this protein.
A comparison of highly purified C'1 esterase inhibitor from human serum and alpha 2-neuraminoglycoprotein from human plasma by immunofusion, immunoelectrophoresis, and discgel electrophoresis showed them to be antigenically identical.
Affected persons in three families with hereditary angioneurotic edema completely lack naturally occurring serum inhibitor of C′1esterase. This inhibitor is also absent from some younger relatives who have not yet had typical attacks of swelling. Its absence is characteristic of the hereditary form of angioneurotic edema, for those with nonhereditary types have normal or increased amounts of the inhibitor. Of 500 persons tested, no others completely lacked this inhibitor.
Immunochemical and enzymatic estimation of C'l esterase inhibitor showed a correlation between the values for normal sera and heparin plasma but not for EDTA plasma. With the enzymatic method, EDTA plasma showed lower values than those obtained with sera and heparin plasma. Low values were found in 14 of 17 patients with hereditary angioneurotic edema. In 3 of these sera the values obtained with the two techniques disagreed; all 3 showed low inhibiting capacity but elevated or normal content of inhibitor protein.
A comparison of highly purified C'1 esterase inhibitor from human serum and alpha(2)-neuraminoglycoprotein from human plasma by immunofusion, immuneelectrophoresis, and discgel electrophoresis showed them to be antigenically identical.
A technique is described which allows direct visualization of individual proteins in mixtures by specific antiserum after electrophoresis. By minimizing diffusion, it permits rapid, direct, and clear detection of genetic polymorphism and ‘conversion’ of proteins in the complement and coagulation systems.
A sensitive technique for the measurement of the activity of the α-2-globulin inhibitor of the activated first component of complement has been described. The reaction is based on the ability of this inhibitor to block C′1a in a stoichiometric fashion in immune hemolysis.
A hemolytic unit of C′1aINH has been defined as the amount required to inhibit 63% of one effective molecule of C′1a in the presence of a total dose of from 1.5 to 2.5 effective molecules per cell. The C′1a inhibitor is stoichiometrically depleted by its interaction with fluid phase hemolytically active C′1a. C′1aINH is equally effective in blocking C′1a in the fluid phase or on the cell.
C′1aINH activity can be measured in human serum after removal of C′1 by precipitating the latter at low ionic strength, pH 7.5. Normal levels for C′1aINH activity are 37,445 ± 17,790 units/ml. Patients with hereditary angioedema were found to have a 99% reduction in C′1aINH activity.
A method is deseribed for quantitative analysis of proteins with a charge differing from that of the bulk of the immunoglobulins. The method utilizes the difference between the rate of electrophoretic migration of proteins and of their antibody complexes in agarose gel.The method has an error of a few per cent; it is rapid, suitable for serial analysis, and requires 2 to 0.5 μg protein antigen. The amount of antiserum needed is slightly less than with the radial immunodiffusion techniques.
IN the methods of iodination currently used only the cationic portion of the iodine molecule becomes bound to the ring structure of tyrosine, so that the theoretical efficiency of labelling is 50 per cent. In practice, efficiencies are always lower than this and may be only a few per cent when the ratio of iodine to protein used is less than one atom per molecule. Values greater than 50 per cent can be obtained by adding oxidizing agents to liberate iodine from iodide, but most if not all of these appear to affect adversely the properties of the labelled protein.
This chapter discusses the different aspects of thiobarbituric acid assay of sialic acid. Periodate oxidation of the neuraminic acid backbone of sialic acids results in the formation of β-formylpyruvic acid from carbon atoms 1 to 4. The N-acetyl or N-glycolyl group of sialic acids apparently does not interfere with periodate oxidation. β-Formylpyruvic acid is coupled with 2-thiobarbituric acid to form a red chromophore with a maximum absorption at 549 mμ. It is found that as only free sialic acids are reactive in the assay, hydrolysis of sialic acid-containing material must be carried out for the measurement of total sialic acids. The assay is suitable for measuring the release of bound sialic acid by sialidase. A series of 2-keto, 3-deoxy sugar acids, found in bacteria, also react in the thiobarbituric acid assay. These produce a chromogen with a peak at 549 mμ. They can be readily distinguished from sialic acids because they are not reactive in the orcinol or direct Ehrlich assays for sialic acids. The hydrolysis frees all the sialic acids from several mucoproteins that have been tested except for brain tissue where release of sialic acids takes place for several hours.
Affected persons in three families with hereditary angioneurotic edema completely lack naturally occurring serum inhibitor of C′1esterase. This inhibitor is also absent from some younger relatives who have not yet had typical attacks of swelling. Its absence is characteristic of the hereditary form of angioneurotic edema, for those with nonhereditary types have normal or increased amounts of the inhibitor. Of 500 persons tested, no others completely lacked this inhibitor. This deficiency did not result from a destructive property in these serum specimens, for normal serum lost no inhibitor during its incubation with that from a patient. In addition, lack of this inhibitor did not reflect a generalized absence of antiproteolytic activity, for antiplasmin and antitrypsin were present. This evidence suggests that absence of serum inhibitor of C′1-esterase is an inherited abnormality in those with the familial type of angioneurotic edema. This does not explain the sporadic occurrence of localized swellings; tissue mechanisms are implicated. This determination may permit identification of young family members who will have attacks of swelling later in life.
Serums of patients with hereditary angioneurotic edema lack inhibitory activity against the esterase derived from the first component of complement. In one group of patients this lack appears to result from failure to synthesize the esterase inhibitor of the first component of complement, whereas in another group of patients an abnormal, nonfunctional protein is synthesized.
Hereditary genetic variants
- F Rosen
Rosen, F. aldson. 1965. Hereditary genetic variants. Science (Washington). 148: 957